Composition of docetaxel liposomal injection with high drug loading

ABSTRACT

The present invention relates to a pharmaceutical liposomal composition comprising of about 0.8% w/w to about 1% w/w of docetaxel, about 30% w/w to about 38% w/w of Soya Phosphatidyl Choline, about 0.2% w/w to about 0.8% w/w of Sodium Cholesteryl Sulfate, about 61% w/w to about 68% w/w of Sucrose and a pH adjusting agent, wherein the pH of liposomal composition is less than 3.5 and the process for preparation thereof.

FIELD OF THE INVENTION

The present invention relates to Docetaxel liposomes for injection whichwill be used to target the tumour site. It relates to increase thedrug's residence time in blood. The present invention also includes toachieve more than 90% drug loading using combination of varioussolvents.

BACKGROUND OF THE INVENTION

Docetaxel is an antineoplastic agent belonging to the taxoid familybeing marketed by Sanofi-Aventis under trade name Taxotere®. It isprepared by semi synthesis beginning with a precursor extracted from therenewable needle biomass of yew plants. The chemical name for docetaxelis (2R,3S)—N-carboxy-3-phenylisoserine, N-tert-butyl ester, 13-esterwith 5beta-20-epoxy-1,2α, 4,7β,10β,13α-hexahydroxytax-11-en-9-one4-acetate 2-benzoate, trihydrate. Docetaxel has the following structuralformula:

Docetaxel binds to free tubulin and promotes the assembly ofmicrotubules, which reduces the availability of tubulin for, and therebyprevents, cell division. Simultaneously, docetaxel inhibits microtubuledisassembly, causing apoptosis. See Taxotere® Prescribing Information.

Docetaxel is marketed as Taxotere®, which is FDA-approved for breastcancer, non-small cell lung cancer, hormone refractory prostate cancer,gastric adenocarcinoma, and squamous cell carcinoma of head and neckcancer. Taxotere is available as a sterile solution of docetaxel in asealed vial, i.e., a single-vial injection concentrate, containing 20mg/mL docetaxel; 0.54 g/mL polysorbate 80, and 0.395 g/mL dehydratedalcohol solution. For administration to patients, an amount of Taxotereinjection concentrate is injected into a 250 mL infusion bag or bottleof either 0.9% sodium chloride solution or 5% dextrose solution toproduce a final concentration of 0.3 to 0.74 mg/ml. The recommendedtherapy is six cycles of docetaxel given once every three weeks. See id.

The presence of polysorbate 80 in Taxotere, however, most often resultsin serious side effects. Such reactions characterized by generalizedrash/erythema, hypotension and/or bronchospasm, or very rarely fatalanaphylaxis, have been reported in patients. Hypersensitivity reactionsrequire immediate discontinuation of the Taxotere infusion andadministration of appropriate therapy.

In order to reduce the side effects induced by polysorbate 80, patientsare treated with dexamethasone for three days prior to therapy.Dexamethasone is a steroid that suppresses the immune response inpatients, which can be especially detrimental in cancer patients underchemotherapy, whose immunity may already be compromised due to thedestruction of healthy cells by the chemotherapeutic treatment. As aresult, these patients can be susceptible to bacterial and fungalinfections. Further, despite receiving the recommended 3-daydexamethasone premedication, patients still report hypersensitivity sideeffects from Taxotere.

Due to these side effects, most of the patients stop Taxotere therapy bythe end of the second or third cycle, skip a dose, or continue furthertherapy at a reduced dose. Similarly, other solubilizing agents such asCREMOPHOR EL®, which is a polyethoxylated castor oil used in connectionwith the marketed paclitaxel product TAXOL®, induce similar allergicreactions requiring premedication with a steroid.

Recently, D-a-tocopheryl PEG 1000 succinate (TPGS), derivative ofvitamin E has been successfully utilized in numerous drug carrierformulations like micro emulsions, micelles, glycerosomes, nanoparticlesand solid dispersions. It is a FDA approved excipient with hydrophilicpolar head and hydrophobic non polar alkyl chain prepared byesterification of vitamin E by polyethylene glycol of molecular weight1000 D. Principally, it is used as solubilizer, emulsifier and vehiclein lipidic formulations. The recent application of TPGS includes oralabsorption enhancer owing to P-gp efflux inhibition, promoting celluptake and drug absorption. Furthermore, TPGS provide better membranestabilizing effect to the lipid bilayer as compared to PEGs owing tobetter hydrophilic lipophilic balance in the molecule.

U.S. Pat. No. 8,591,942 discloses the method of preparing liposomescontaining docetaxel, the method consisting of dispersing soyphosphatidylcholine and sodium oleate in an aqueous medium to producedispersed liposomes.

U.S. Pat. No. 9,655,846 discloses the suspension of liposomes in anaqueous medium, the liposomes encapsulating and solubilizing docetaxelat a concentration of at least about 5 mg docetaxel per ml of theaqueous medium.

U.S. Pat. No. 8,912,228 discloses the sterile pharmaceutical formulationfor use in treatment of a patient in need thereof, comprising docetaxelor a pharmaceutically acceptable salt thereof, one or more solubilizers,a-lipoic acid, TPGS, one or more hydrotropes, and optionally one or moreagents having a pK_(a) of about 3 to about 6.

US Application No. US20080166403 discloses the long circulatingliposome, comprising a phospholipid bilayer and a hydrophilic core,wherein the phospholipid bilayer contains vitamin E derivative (D-alphatocopheryl polyethylene glycol 1000 succinate, TPGS).

PCT Patent Publication No. WO2014167435A2 discloses the surfacefunctionalized liposomal formulation comprising an anticancer agent asan active ingredient, liposomes surrounded by a functional coating ofD-a-Tocopheryl Polyethylene Glycol 1000 Succinate (TPGS), wherein theanticancer agent is entrapped within the liposomes, and further whereinsaid formulation has an encapsulation efficiency of >70%.

CN Patent No. 101991538 discloses the use of a TPGS-containing liposomecomposition in preparation of drug-loaded liposomes.

Muthu et al.; Biomaterials. 2012 April; 33(12):3494-501 recentlydisclosed the TPGS coated liposomes for brain delivery of docetaxelprepared by solvent injection method. The reported formulations posedabout 64.10±0.57% encapsulation efficiency which was significantly lowerthan the present invention (encapsulation efficiency ˜83.63±1.16%). Theprobable reasons for this appreciation of encapsulation efficiency inthe present invention could be attributed to the exhaustive optimizationof the various process parameters, composition of excipients and methodof preparation of the liposomes. The same group in further extension ofwork improved the therapeutic efficacy of the docetaxel by combinationtherapy with quantum dots. However, the present invention does notinculcate any such combination therapy.

However, there exists a need to develop a docetaxel liposomal injectionformulation is needed to avoid these side effects, premedicationrequirements, and patient noncompliance issues associated with thecurrently marketed formulation of Taxotere.

SUMMARY OF THE INVENTION

In one object, the present invention provides herein, liposomalinjection compositions consisting essentially of a therapeuticallyeffective amount of docetaxel and an excipient that facilitatesintravenous administration, and which will be used to target the tumoursite. It relates to increase the drug's residence time in blood. Thepresent invention also includes to achieve more than 90% drug loadingusing combination of various solvents, preferably methanol and tertiarybutanol (T-butanol or tertiary butyl alcohol) in ratio of 1:1.

In another object, the present invention further provides the docetaxelliposomal injection consisting essentially of docetaxel, phospholipids,cholesterol, lyoprotectant and a pH adjusting agent.

Definition of Selected Terms

In describing and claiming the present invention, the followingterminologies will be used in accordance with the definitions set outbelow.

The term “liposomes” are vesicles composed of one or more concentriclipid bilayers which contain an entrapped aqueous volume. The bilayersare composed of two lipid monolayers having a hydrophobic “tail” regionand a o hydrophilic “head” region, where the hydrophobic regions orienttoward the center of the bilayer and the hydrophilic regions orienttoward the inner or outer aqueous phase.

As used herein in connection with numerical values, the terms “about”mean+/−10% of the indicated value, including the indicated value.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides herein, compositions consistingessentially of a therapeutically effective amount of docetaxel and anexcipient that facilitates intravenous administration, and which will beused to target the tumour site. It relates to increase the drug'sresidence time in blood. The present invention also includes to achievemore than 90% drug loading using combination of various solvents,preferably methanol and tertiary butanol (T-butanol or tertiary butylalcohol) in ratio of 1:1.

In a preferred embodiment, the pharmaceutical composition of theinvention is liposomal injection.

In the most preferred embodiment, the pharmaceutical liposomal injectioncomposition comprises docetaxel and pharmaceutically acceptableexcipients.

Docetaxel, preferably used in the present pharmaceutical liposomalcomposition is of about 0.8% w/w to about 1% w/w based on the totalweight of the composition. The preferred concentration of docetaxel incomposition is about 0.85% w/w to about 0.95% w/w based on the totalweight of the composition. Most preferably, the docetaxel is used in thecomposition of about 0.9% w/w based on total weight of the composition.

In further embodiment, the pharmaceutical liposomal compositioncomprises docetaxel, phospholipids, cholesterol, lyoprotectant and a pHadjusting agent, wherein the pH of liposomal composition is less than3.5.

In yet another embodiment, the present invention further provides thedocetaxel liposomal composition comprising docetaxel, phospholipids,cholesterol, solubilizer, lyoprotectant, and a pH adjusting agent.

Examples of the phospholipids are selected form the group consisting ofa natural phospholipid, a synthetic phospholipid, and combinationsthereof. Lecithin is one of the natural resources for phospholipid.Lecithin is a mixture found in egg yolk and soya. It comprises a numberof phospholipids including phosphatidylcholine (PC, Soya PhosphatidylCholine), phosphatidylethanolamine (PE), and phosphatidylinositol (PI).Natural phospholipids also include, e.g. soy phosphatidyl choline (SPC),sphingomyelin, and phosphatidylglycerol (PG). Synthetic phospholipidsinclude, but are not limited to, derivatives of phosphocholine (forexample, DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC), derivatives ofphosphoglycerol (for example, DMPG, DPPG, DSPG, POPG, DSPG-NA,DSPG-NH4), derivatives of phosphatidic acid (for example, DMPA, DPPA,DSPA), derivatives of phosphoethanolamine (for example, DMPE, DPPE, DSPEDOPE), derivatives of phosphoserine (for example, DOPS), PEG derivativesof phospholipid (for example, mPEG-phospholipid, mPEG 2000-DSPE,polyglycerin-phospholipid, functionalized-phospholipid, and terminalactivated-phospholipid) and any mixtures thereof. Preferably,phospholipid is selected from soy phosphatidyl choline (SPC) and mixturephospholipids are selected from soy phosphatidyl choline (SPC) andN-Carbonylmethoxypolyethylenglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE).Phospholipids preferably used in the pharmaceutical liposomalcomposition of present invention is Soya Phosphatidyl Choline. SoyaPhosphatidyl Choline used in the present invention is from about 30% w/wto about 40% w/w based on the total weight of the composition,preferably from about 30% w/w to about 38% w/w based on the total weightof composition, even more preferably, of about 31% w/w to about 36% w/wbased on total weight of the composition and most preferably of about32% w/w based on the total weight of the composition.

Examples of the cholesterol is selected from the group consisting ofcholesterol, cholesteryl sulfate and its salts (e.g., sodium salt),cholesteryl hemisuccinate, cholesteryl succinate, cholesteryl oleate,polyethylene glycol derivatives of cholesterol (cholesterol-PEG),coprostanol, cholestanol, cholestane, cholic acid, cortisol,corticosterone, hydrocortisone and calciferol. Preferably, thecholesterol is selected from Sodium Cholesteryl Sulfate. SodiumCholesteryl sulfate is preferably used in the range from about 0.2% w/wto about 0.8% w/w based on the total weight of the composition, morepreferably, of about 0.4% w/w to about 0.6% w/w based on total weight ofthe composition, and most preferably of about 0.5% w/w based on totalweight of composition.

Examples of the solubilizer is selected from the group consistingvitamin E TPGS, polyethylene glycol (PEG) 400, and propylene glycol (PG)tween 80, tween 20, glycerol span 80 and glycofurol. Preferably, thesolubilizer is selected from vitamin E TPGS. Solubilizer preferably usedin the pharmaceutical liposomal injection composition of about 0.1% toabout 1.5% based on the total weight of the composition.

Examples of the lyoprotectant are selected from the group consisting ofsucrose, trehalose, arabinose, erythritol, fructose, galactose, glucose,lactose, maltitol, maltose, maltotriose, mannitol, mannobiose, mannose,ribose, sorbitol, saccharose, xylitol, xylose, dextran, or a mixturethereof. Preferably, the lyoprotectant is selected from sucrose. Sucrosepreferably used in the pharmaceutical liposomal composition is about 61%to about 68% based on the total weight of the composition, morepreferably of about 62% to about 66% based on total weight of thecomposition and most preferably of about 66.5% w/w based on the totalweight of composition.

Examples of pH adjusting agents used in the present liposomalcomposition is hydrochloric acid. pH adjusting agent is used to adjustthe pH of the liposomal composition to about less than pH 3.5, morepreferably pH of about 3. The inventors of the present invention havesurprisingly found that with the pH of less than 3.5, there was highdrug loading of more than 90% docetaxel into liposomes (more 90% ofdocetaxel is encapsulated into liposomes as bound drug and less thanabout 10% of docetaxel is present as free drug).

In embodiments of the present invention, the present invention providesa pharmaceutical liposomal composition comprising of about 0.8% w/w toabout 1% w/w of docetaxel, about 30% w/w to about 38% w/w of SoyaPhosphatidyl Choline, about 0.2% w/w to about 0.8% w/w of SodiumCholesteryl Sulfate, about 61% w/w to about 68% w/w of Sucrose and a pHadjusting agent, wherein the pH of liposomal composition is less than3.5.

In further embodiment of the present invention, the present inventionprovides a pharmaceutical liposomal composition consisting essentiallyof about 0.8% w/w to about 1% w/w of docetaxel, about 30% w/w to about38% w/w of Soya Phosphatidyl Choline, about 0.2% w/w to about 0.8% w/wof Sodium Cholesteryl Sulfate, about 61% w/w to about 68% w/w of Sucroseand a pH adjusting agent, wherein the pH of liposomal composition isless than 3.5.

In another embodiment of the present invention, the present inventionprovides a pharmaceutical liposomal composition consisting of about 0.8%w/w to about 1% w/w of docetaxel, about 30% w/w to about 38% w/w of SoyaPhosphatidyl Choline, about 0.2% w/w to about 0.8% w/w of SodiumCholesteryl Sulfate, about 61% w/w to about 68% w/w of Sucrose and a pHadjusting agent, wherein the pH of liposomal composition is less than3.5.

In specific embodiment, the present invention provides a pharmaceuticalliposomal composition comprising of about 0.9% w/w of docetaxel, about32% w/w of Soya Phosphatidyl Choline, about 0.5% w/w of SodiumCholesteryl Sulfate, about 66.5% w/w of Sucrose and a pH adjustingagent, wherein the pH of liposomal composition is about 3.

In another embodiment, the present invention provides a pharmaceuticalliposomal composition consisting essentially of about 0.9% w/w ofdocetaxel, about 32% w/w of Soya Phosphatidyl Choline, about 0.5% w/w ofSodium Cholesteryl Sulfate, about 66.5% w/w of Sucrose and a pHadjusting agent, wherein the pH of liposomal composition is about 3.

In further embodiment, the present invention provides a pharmaceuticalliposomal composition consisting essentially of about 0.9% w/w ofdocetaxel, about 32% w/w of Soya Phosphatidyl Choline, about 0.5% w/w ofSodium Cholesteryl Sulfate, about 66.5% w/w of Sucrose and a pHadjusting agent, wherein the pH of liposomal composition is about 3.

In specific embodiment, the present invention provides a pharmaceuticalliposomal composition comprising of about 0.9% w/w of docetaxel, about32% w/w of Soya Phosphatidyl Choline, about 0.5% w/w of SodiumCholesteryl Sulfate, about 66.5% w/w of Sucrose and a pH adjustingagent, wherein the pH of liposomal composition is of about 2.5 to about3.2.

In another embodiment, the present invention provides a pharmaceuticalliposomal composition consisting essentially of about 0.9% w/w ofdocetaxel, about 32% w/w of Soya Phosphatidyl Choline, about 0.5% w/w ofSodium Cholesteryl Sulfate, about 66.5% w/w of Sucrose and a pHadjusting agent, wherein the pH of liposomal composition is of about 2.5to about 3.2.

In further embodiment, the present invention provides a pharmaceuticalliposomal composition consisting essentially of about 0.9% w/w ofdocetaxel, about 32% w/w of Soya Phosphatidyl Choline, about 0.5% w/w ofSodium Cholesteryl Sulfate, about 66.5% w/w of Sucrose and a pHadjusting agent, wherein the pH of liposomal composition is of about 2.5to about 3.2.

The pharmaceutical liposomal composition of present invention comprisesthe liposomes of d₉₀ less than 200 nm, d₅₀ less than 150 nm and d₁₀ lessthan 100 nm.

The docetaxel liposomes of the present invention are prepared by aprocess comprising the steps of comprising the steps of:

-   -   a. dispersing Soya Phosphatidyl Choline in solvent mixture to        solubilize Soya Phosphatidyl Choline;    -   b. adding sodium cholesteryl sulfate to the solubilized Soya        Phosphatidyl Choline;    -   c. adding docetaxel to contents of step b;    -   d. preparing sucrose solution by dissolving sucrose in purified        water and adding the pH adjusting agent to form the sucrose        solution, wherein the pH of sucrose solution is about 3.    -   e. adding contents of step c to step d and mixing with high hear        at 8000 RPM for 15 minutes    -   f. rota evaporation;    -   g. addition of pH adjusting agent to pH of about 3;    -   h. extrusion of liposomes containing docetaxel to the particle        size d₉₀ of less than 200 nm;    -   i. filtration and    -   j. lyophilization.

Examples of the solvents are selected from the group consisting ofmethanol, ethanol (anhydrous alcohol), propanol, butanol (t-butanol,tertiary butyl alcohol), chloroform, isoamyl alcohol, isopropanol,2-methoxy ethanol, Tetrahydrofuran, DMSO acetone, acetonitrile and anycombinations thereof. The solvents preferably used for the preparationof liposomal composition is tertiary butyl alcohol and methanol in theratio of 1:1.

In a preferred embodiment, the present invention relates to the methodof preparing liposomal composition comprising the steps of:

-   -   a. dispersing Soya Phosphatidyl Choline in solvent mixture of        methanol and tertiary butyl alcohol to solubilize Soya        Phosphatidyl Choline;    -   b. adding sodium cholesteryl sulfate to the solubilized Soya        Phosphatidyl Choline;    -   c. adding docetaxel to contents of step b;    -   d. preparing sucrose solution by dissolving sucrose in purified        water and adding the pH adjusting agent to form the sucrose        solution, wherein the pH of sucrose solution is about 3.    -   e. adding contents of step c to step d and mixing with high hear        at 8000 RPM for 15 minutes    -   f. rota evaporation;    -   g. addition of pH adjusting agent to pH of about 3;    -   h. extrusion of liposomes containing docetaxel to the particle        size d₉₀ of less than 200 nm;    -   i. filtration and    -   j. lyophilization.

In a more preferred embodiment, the present invention relates to themethod of preparing liposomal composition comprising the steps of:

-   -   a. dispersing Soya Phosphatidyl Choline in solvent mixture of        methanol and tertiary butyl alcohol in the ratio of 1:1 to        solubilize Soya Phosphatidyl Choline;    -   b. adding sodium cholesteryl sulfate to the solubilized Soya        Phosphatidyl Choline;    -   c. adding docetaxel to contents of step b;    -   d. preparing sucrose solution by dissolving sucrose in purified        water and adding the pH adjusting agent to form the sucrose        solution, wherein the pH of sucrose solution is about 3.    -   e. adding contents of step c to step d and mixing with high hear        at 8000 RPM for 15 minutes    -   f. rota evaporation;    -   g. addition of pH adjusting agent to pH of about 3;    -   h. extrusion of liposomes containing docetaxel to the particle        size d₉₀ of less than 200 nm;    -   i. filtration and    -   j. lyophilization.

In another embodiment liposomal docetaxel liquid filtrate is lyophilizedby comprising the steps of freezing the filtrate at temperature rangingfrom about −5° C. to about −50° C. for the time duration ranging fromabout 10 hours to about 20 hours; drying under vacuum at a temperatureranging from about −50° C. to about 40° C. for time duration rangingfrom about 40 hours to about 80 hours.

In a further specific embodiment liposomal docetaxel liquid filtrate islyophilized by comprising the steps of

-   -   a. Loading the filtrate filled vials at −5° C.±2° C.;    -   b. Freezing the filtrate formulation at −5° C.±2° C. for 100        minutes±20 minutes    -   c. Maintaining the freezing temperature for another 300        minutes±20 minutes    -   d. Reducing the temperature up to −25° C.±2° C. for 50        minutes±10 minutes    -   e. Maintaining the reduced temperature for another 90 minutes±10        minutes    -   f. Reducing the temperature up to −50° C.±2° C. for 60        minutes±10 minutes    -   g. Maintaining the reduced temperature for another 300        minutes±10 minutes    -   h. Evacuating the filtrate by creating vacuum of 750 m Torr to        obtain frozen formulation    -   i. Drying the frozen formulation at −50° C.±2° C. by creating        vacuum at 750 m Torr for 30 minutes±10 minutes    -   j. Drying the frozen formulation at −35° C.±2° C. by creating        vacuum at 400 m Torr for 120 minutes±10 minutes    -   k. Maintaining the drying for another 1255 minutes±20 minutes at        −35° C.±2° C. and 400 m Torr vacuum.    -   l. Drying the frozen formulation at −25° C.±2° C. by creating        vacuum at 300 m Torr for 150 minutes±10 minutes    -   m. Maintaining the drying for another 600 minutes±20 minutes at        −25° C.±2° C. and 300 m Torr vacuum.    -   n. Drying the frozen formulation at −5° C.±2° C. by creating        vacuum at 200 m Torr for 150 minutes±10 minutes    -   o. Maintaining the drying for another 900 minutes±20 minutes at        −5° C.±2° C. and 200 m Torr vacuum.    -   p. Drying the frozen formulation at 20° C.±2° C. by creating        vacuum at 100 m Torr for 150 minutes±10 minutes    -   q. Maintaining the drying for another 300 minutes±20 minutes at        20° C.±2° C. and 100 m Torr vacuum.    -   r. Drying the frozen formulation at 25° C.±2° C. by creating        vacuum at 100 m Torr for 30 minutes±10 minutes    -   s. Maintaining the drying for another 150 minutes±20 minutes at        25° C.±2° C. and 100 m Torr vacuum.    -   t. Drying the frozen formulation at 40° C.±2° C. by creating        vacuum at 100 m Torr for 30 minutes±10 minutes    -   u. Maintaining the drying for another 120 minutes±20 minutes at        40° C.±2° C. and 100 m Torr vacuum    -   v. Drying the frozen formulation at 25° C.±2° C. by creating        vacuum at 100 m Torr for 30 minutes±10 minutes    -   w. Maintaining the drying for another 60 minutes±20 minutes at        25° C.±2° C. and 100 m Torr vacuum.

In the embodiments of the present invention, the lyophilized compositionof present invention is administered intravenously to the patients forthe treatment of breast cancer, Non-small cell lung cancer, Castrationresistant prostate cancer, gastric adenocarcinoma and Squamous CellCarcinoma of the Head and Neck Cancer.

In embodiments of the invention for intravenous administration, thelyophilized composition is reconstituted with purified water and furtherdiluted with either 0.9% sodium chloride solution or 5% dextrosesolution.

In embodiments of the invention the docetaxel liposomal composition ofpresent invention is used for the treatment of breast cancer, Non-smallcell lung cancer, Castration resistant prostate cancer, gastricadenocarcinoma and Squamous Cell Carcinoma of the Head and Neck Cancer,wherein the pre-medication with prednisone is not required.

In embodiments of the invention the recommended dose of liposomalcomposition of present invention is 60 mg/m² to 100 mg/m² administeredintravenously over 1 hour every 3 weeks.

The following examples are provided to illustrate the present invention.It is understood, however, that the invention is not limited to thespecific conditions or details described in the examples below. Theexamples should not be construed as limiting the invention as theexamples merely provide specific methodology useful in the understandingand practice of the invention and its various aspects. While certainpreferred and alternative embodiments of the invention have been setforth for purposes of disclosing the invention, modification to thedisclosed embodiments can occur to those who are skilled in the art.

Examples 1 to 3

Liposomal injection with the following compositions are prepared.

Example 1 Example 2 Example 3 Ingredients (% w/w) (% w/w) (% w/w)Docetaxel anhydrous 0.84%-1%   0.84%-0.87% 0.84%-0.87% Soya PhosphatidylCholine 30%-38% 30%-38% 30%-38% Sodium Cholesteryl Sulfate 0.2%-0.8%0.2%-0.8% 0.2%-0.8% MPEG2000-DSPE — 0.5%-2%   — (N-(Carbonylmethoxypoly- ethylenglycol-2000)-1,2- distearoyl-sn-glycero-3-phosphoethanolamine) Vitamin E TPGS — — 0.5%-1.5% Sucrose 61%-68%61%-68% 61%-68% Solvent mixture Q.S Q.S Q.S 0.1N Hydrochloric acid asQ.S Q.S Q.S pH adjuster

These Liposomal injections are prepared as follows:

-   -   1. Prepare solvent mixture of solubilization of Lipids and Drug        by mixing 50:50 v/v of Methanol and T-Butanol mixture.    -   2. To 4 mL of the solvent mixture of step 1, add and solubilize,        weighed quantity of Soya Phosphatidyl Choline, at 60-65° C.    -   3. To the Lipid solution of step 2, add and solubilize, weighed        quantity of Sodium Cholesteryl Sulfate, at 60-65° C.    -   4. To the Lipid solution of step 3, add and solubilize, weighed        quantity of MPEG 2000 DSPE/Vitamin E TPGS, at 60-65° C.        (optionally).    -   5. To the Lipid mixture of step 4, add and solubilize, weighed        quantity of Docetaxel at 60-65° C.    -   6. Mix the contents of step 4 for 10 minutes at 60-65° C. for        uniform binding of docetaxel with the lipids.    -   7. Prepare sucrose solution by dissolving the weighed quantity        of sucrose in purified water equivalent to 75% of the batch        size.    -   8. Prepare 0.1 N Hydrochloric acid by diluting the required        quantity of 37% concentrated HCl.    -   9. Adjust the pH of the sucrose solution prepared in the step 7        between pH 3.0 to 4.0 using the 0.1N HCl.    -   10. Heat the Sucrose solution of step 8 to 60-65° C.    -   11. Add the Drug Lipid Mixture to Sucrose solution under High        Shear Mixing at 5000-20000 RPM by Ethanol Injection method and        rinse the container containing Lipid Drug mixture with 1 mL of        Solvent Mixture and run the mixture for 2 to 30 minutes.    -   12. Volume was made up to the mark of the liposomal formulation        of step 11, with purified water and if required pH was adjusted        to between 3.0 to 4.0.    -   13. Size reduction of liposomal formulation of step 12, was done        using extrusion with 400 nm, 200 nm, 100 nm and 50 nm        polycarbonate filters.    -   14. Post size reduction, the liposomal formulation is sterile        filtered using 0.22-micron filter.    -   15. Post sterile filtration, the samples are freeze dried to get        the dry liposomal cake for Injection.

Example 4

Ingredients Example 4 (% w/w) Docetaxel anhydrous 0.91 Soya PhosphatidylCholine 32.05 Sodium Cholesteryl Sulfate 0.5 Sucrose 66.54 Methanol Q.STertiary butyl alcohol Q.S 0.1N Hydrochloric acid as pH adjuster Q.S topH 3 Purified water Q.SProcess for Preparation of Liposomes Encapsulated with Docetaxel:

-   -   1. 14.46 g (32.05% w/w) of soya phosphatidyl choline was        dispersed in 8 mL of solvent mixture (4 mL of tertiary butyl        alcohol and 4 mL of methanol in ratio of 1:1) and mixed with        magnetic stirrer in water bath at 47° C. for 25 minutes to        solubilize soya phosphatidyl choline.    -   2. To the solubilized sodium phosphatidyl choline solution of        step 1, 226.6 mg (0.5% w/w) of sodium cholesteryl sulfate was        added and solubilised with magnetic stirrer in water bath at        49° C. for 45 minutes to form the dispersed liposomes.    -   3. To the contents of step 3, 412.1 mg (0.91% w/w) of solid        docetaxel anhydrous was added and solubilized with magnetic        stirrer in water bath at 46° C. for 10 minutes to form docetaxel        containing dispersed liposomes.    -   4. The contents of step 3, as added to sucrose solution (sucrose        solution was prepared by dissolving 30.02 g [66.54% w/w] of        sucrose in required quantity of purified water with magnetic        stirrer in water bath at 45° C. for 2 minutes and further pH is        adjusted to 2.7 using 0.1N hydrochloric acid solution) using        high shear mixing ultra-turrax T-25 digital at 8000 RPM for 15        minutes.    -   5. The contents of step 4, was subjected to rota evaporation        with chiller temperature of 2° C., bath temperature of 46° C.        with vacuum.    -   6. After rota evaporation, to contents of step 5, required        quantity of purified water was added and pH was adjusted to 3        using 0.1N hydrochloric acid solution.    -   7. The contents of step 6, was extruded with 200 nm, 100 nm, 80        nm and 50 nm polycarbonate membranes using lipex extruder at        47° C. Three cycles of passing resulted in a liposome with        particle size d90 of less than 200 nm (i.e 178 nm), d50 of less        than 150 nm (i.e 113 nm) and d10 of less than 100 nm (i.e 73 nm)    -   8. The contents of step 7, was filtered using 0.2 μm membrane        filter.    -   9. The filtrate of step 8, was filled into 30 mL moulded vials        and lyophilized using the following lyo cycle.        -   a. Loading the filtrate filled vials at −5° C.±2° C.;        -   b. Freezing the filtrate formulation at −5° C.±2° C. for 100            minutes±20 minutes        -   c. Maintaining the freezing temperature for another 300            minutes±20 minutes        -   d. Reducing the temperature up to −25° C.±2° C. for 50            minutes±10 minutes        -   e. Maintaining the reduced temperature for another 90            minutes±10 minutes        -   f. Reducing the temperature up to −50° C.±2° C. for 60            minutes±10 minutes        -   g. Maintaining the reduced temperature for another 300            minutes±10 minutes        -   h. Evacuating the filtrate by creating vacuum of 750 m Torr            to obtain frozen formulation        -   i. Drying the frozen formulation at −50° C.±2° C. by            creating vacuum at 750 m Torr for 30 minutes±10 minutes        -   j. Drying the frozen formulation at −35° C.±2° C. by            creating vacuum at 400 m Torr for 120 minutes±10 minutes        -   k. Maintaining the drying for another 1255 minutes±20            minutes at −35° C.±2° C. and 400 m Torr vacuum.        -   l. Drying the frozen formulation at −25° C.±2° C. by            creating vacuum at 300 m Torr for 150 minutes±10 minutes        -   m. Maintaining the drying for another 600 minutes±20 minutes            at −25° C.±2° C. and 300 m Torr vacuum.        -   n. Drying the frozen formulation at −5° C.±2° C. by creating            vacuum at 200 m Torr for 150 minutes±10 minutes        -   o. Maintaining the drying for another 900 minutes±20 minutes            at −5° C.±2° C. and 200 m Torr vacuum.        -   p. Drying the frozen formulation at 20° C.±2° C. by creating            vacuum at 100 m Torr for 150 minutes±10 minutes        -   q. Maintaining the drying for another 300 minutes±20 minutes            at 20° C.±2° C. and 100 m Torr vacuum.        -   r. Drying the frozen formulation at 25° C. 2° C. by creating            vacuum at 100 m Torr for 30 minutes±10 minutes        -   s. Maintaining the drying for another 150 minutes±20 minutes            at 25° C.±2° C. and 100 m Torr vacuum.        -   t. Drying the frozen formulation at 40° C.±2° C. by creating            vacuum at 100 m Torr for 30 minutes±10 minutes        -   u. Maintaining the drying for another 120 minutes±20 minutes            at 40° C.±2° C. and 100 m Torr vacuum        -   v. Drying the frozen formulation at 25° C.±2° C. by creating            vacuum at 100 m Torr for 30 minutes±10 minutes        -   w. Maintaining the drying for another 60 minutes±20 minutes            at 25° C.±2° C. and 100 m Torr vacuum.

Comparative Example 1

Ingredients Comparative Example 1 (% w/w) Docetaxel anhydrous 0.91 SoyaPhosphatidyl Choline 32.05 Sodium Cholesteryl Sulfate 0.5 Sucrose 66.54Methanol Q.S Ethanol Q.S 0.1N Hydrochloric acid as pH adjuster Q.S to pH4 Purified water Q.S

The process for preparation is same as that of Example 4, with thechanges in solvent mixture of Methanol and Ethanol in ratio of 1:1, pHof 4, without rota evaporation process (step 5) and change inlyophilization cycle (without 40° C. drying step t and u).

Comparative Example 2

Ingredients Comparative Example 2 (% w/w) Docetaxel anhydrous 0.91 SoyaPhosphatidyl Choline 32.05 Sodium Cholesteryl Sulfate 0.5 Sucrose 66.54Methanol Q.S Tertiary butyl alcohol Q.S 0.1N Hydrochloric acid as pHadjuster Q.S to pH 4.5 Purified water Q.S

The process for preparation is same as that of Example 4, with thechanges in pH of formulation adjusted to 4.5

Comparative Example 3

Ingredients Comparative Example 3 (% w/w) Docetaxel anhydrous 0.91 SoyaPhosphatidyl Choline 32.05 Sodium Cholesteryl Sulfate 0.5 Sucrose 66.54Methanol Q.S Tertiary butyl alcohol Q.S 0.1N Hydrochloric acid as pHadjuster Q.S to pH 3 Purified water Q.S

The process for preparation is same as that of Example 4, with change inlyophilization cycle (without 40° C. drying step t and u).

Example 5

Free Drug, Entrapped Drug, Assay, pH, Residual Solvents of Example-4,Comparative Example 1, 2 & 3.

Comparative Comparative Comparative Test Example 4 Ex. 1 Ex. 2 Ex. 3Free Drug 11.1% 29.1% 56.3% 8.8% Entrapped Drug 94.8% 63.7% 47.3% 88.1%Assay 103.6% 93.5% 103.3% 96.0% pH 3.1 4.1 4.7 3 Residual SolventsMethanol 384 ppm — — 1766 ppm T-Butanol 741 ppm — — 5672 ppm Ethanol — —— —

For the measurement of pH, the lyophilized vial of inventive example 4was reconstituted with purified water to produce 2 mg/mL liposomalformulation of docetaxel.

The Free Drug, Entrapped Drug, Assay of docetaxel liposomal formulationwas performed by HPLC and Residual Solvent analysis was performed by Gaschromatography as per the available literature to the personal skilledin the art.

The inventors of present invention have surprisingly found that theexample 4 formulation has high drug loading efficiency (about 95%) withthe solvents of methanol and tertiary butanol in the ratio of 1:1, rotaevaporated, with the formulation pH of about 3 and further the residualsolvents (methanol and tertiary butanol) are less (within the limits oflCH) in comparision to comparative example 1 (containing solvent mixtureof ethanol and methanol in ratio of 1:1 at pH of 4.1), comparativeexample 2 (containing methanol and T-butanol in ratio of 1:1 at pH of4.7) and comparative example 3 (containing methanol and T-butanol inratio of 1:1 at pH of 3, without the drying step at 40° C. inlyophilization step t and u of example 4).

The invention claimed is:
 1. A pharmaceutical liposomal compositioncomprising about 0.8% w/w to about 1% w/w of docetaxel, about 30% w/w toabout 38% w/w of soya phosphatidyl choline, about 0.2% w/w to about 0.8%w/w of sodium cholesteryl sulfate, about 61% w/w to about 68% w/w ofsucrose and a pH adjusting agent, wherein the pH of liposomalcomposition is less than 3.5; wherein the method for preparing theliposomal composition comprises the steps of: (a) dispersing soyaphosphatidyl choline in solvent mixture of methanol and tertiary butylalcohol to solubilize soya phosphatidyl choline; (b) adding sodiumcholesteryl sulfate to the solubilized soya phosphatidyl choline; (c)adding docetaxel to contents of step (b); (d) preparing sucrose solutionby dissolving sucrose in purified water and adding the pH adjustingagent to form the sucrose solution, wherein the pH of sucrose solutionis about 3; (e) adding contents of step (c) to step (d) and mixing withhigh shear at 8000 RPM for 15 minutes; (f) rota evaporation; (g)addition of pH adjusting agent to pH of about 3; (h) extrusion ofliposomes containing docetaxel to the particle size d₉₀ of less than 200nm; (i) filtration and (j) lyophilization.
 2. A method of preparingliposomal composition comprising the steps of: (a) dispersing soyaphosphatidyl choline in solvent mixture of methanol and tertiary butylalcohol to solubilize soya phosphatidyl choline; (b) adding sodiumcholesteryl sulfate to the solubilized soya phosphatidyl choline; (c)adding docetaxel to contents of step (b); (d) preparing sucrose solutionby dissolving sucrose in purified water and adding the pH adjustingagent to form the sucrose solution, wherein the pH of sucrose solutionis about 3; (e) adding contents of step (c) to step (d) and mixing withhigh shear at 8000 RPM for 15 minutes; (f) rota evaporation; (g)addition of pH adjusting agent to pH of about 3; (h) extrusion ofliposomes containing docetaxel to the particle size d₉₀ of less than 200nm; (i) filtration and (j) lyophilization.
 3. The method of claim 2,wherein the solvent mixture of methanol and tertiary butyl alcohol is inthe ratio of 1:1.
 4. The method of claim 2, wherein the lyophilizationof filtrate comprises the steps of freezing the filtrate at temperatureranging from about −5° C. to about −50° C. for the time duration rangingfrom about 10 hours to about 20 hours; drying under vacuum at atemperature ranging from about −50° C. to about 40° C. for time durationranging from about 40 hours to about 80 hours.
 5. The method of claim 2,wherein the lyophilization cycle comprises the steps of (a) loading thefiltrate filled vials at −5° C.±2° C.; (b) freezing the filtrateformulation at −5° C.±2° C. for 100 minutes±20 minutes; (c) maintainingthe freezing temperature for another 300 minutes±20 minutes; (d)reducing the temperature up to −25° C.±2° C. for 50 minutes±10 minutes;(e) maintaining the reduced temperature for another 90 minutes±10minutes; (f) reducing the temperature up to −50° C.±2° C. for 60minutes±10 minutes; (g) maintaining the reduced temperature for another300 minutes±10 minutes; (h) evacuating the filtrate by creating vacuumof 750 m Torr to obtain frozen formulation; (i) drying the frozenformulation at −50° C.±2° C. by creating vacuum at 750 m Torr for 30minutes±10 minutes; (j) drying the frozen formulation at −35° C.±2° C.by creating vacuum at 400 m Torr for 120 minutes±10 minutes; (k)maintaining the drying for another 1255 minutes±20 minutes at −35° C.±2°C. and 400 m Torr vacuum; (l) drying the frozen formulation at −25°C.±2° C. by creating vacuum at 300 m Torr for 150 minutes±10 minutes;(m) maintaining the drying for another 600 minutes±20 minutes at −25°C.±2° C. and 300 m Torr vacuum; (n) drying the frozen formulation at −5°C.±2° C. by creating vacuum at 200 m Torr for 150 minutes±10 minutes;(o) maintaining the drying for another 900 minutes±20 minutes at −5°C.±2° C. and 200 m Torr vacuum; (p) drying the frozen formulation at 20°C.±2° C. by creating vacuum at 100 m Torr for 150 minutes±10 minutes;and (q) maintaining the drying for another 300 minutes±20 minutes at 20°C.±2° C. and 100 m Torr vacuum.